跳到主要內容

臺灣博碩士論文加值系統

(3.231.230.177) 您好!臺灣時間:2021/08/04 05:58
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:謝佶峻
研究生(外文):Hsieh, Chi-Chun
論文名稱:Prostacyclin與PPAR-α啟動劑藉由恢復14-3-3β蛋白保護血管平滑肌細胞的存活
論文名稱(外文):Prostacyclin and PPAR-α agonists protect vascular smooth muscle cell survival by restoring 14-3-3β
指導教授:伍焜玉伍焜玉引用關係
指導教授(外文):Wu, Kun-Yu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:70
中文關鍵詞:血管平滑肌細胞動脈粥狀硬化細胞凋亡
外文關鍵詞:PPAR14-3-3vascular smooth muscle cellatherosclerosisapoptosis
相關次數:
  • 被引用被引用:0
  • 點閱點閱:121
  • 評分評分:
  • 下載下載:16
  • 收藏至我的研究室書目清單書目收藏:0
血管平滑肌細胞(vascular smooth muscle cell,簡稱VSMC)的存活在血管的完整性上扮演極重要的角色,當血管受到損傷所導致VSMC的細胞凋亡(apoptosis),除了會引發其他VSMC的移動(migration)與增生(proliferation)外,更會誘導血管內膜增生(intimal hyperplasia)及血管重新塑造(vascular remodeling),在氧化壓力(oxidative stress)下VSMC的存活機制到目前為止仍未完全被了解。我們假設在血管內的前列腺素(prostacyclin,簡稱PGI2)主要由血管內皮細胞(vascular endothelial cell,簡稱EC)所產生而不是VSMC,而此由EC所產生之PGI2保護VSMC的存活,主要是藉由恢復過氧化體增殖物活化α受體(peroxisome proliferator-activated receptor α,簡稱PPARα)依靠之途徑中的14-3-3蛋白。我們使用能同時大量表現環氧化酶-1(cyclooxygenase-1)與PGI合成酶(prostacyclin synthase)的腺病毒載體(adenoviral vector簡稱Ad-COPI)轉染大鼠VSMC,使得VSMC自行生產大量PGI2,Ad-COPI轉染的VSMC對於H2O2誘導的細胞凋亡有較強的耐受性,同時carbaprostacyclin (簡稱cPGI2)為一種PGI2的穩定類似物,亦會降低H2O2誘導的細胞凋亡。cPGI2的抗細胞凋亡的作用會被PPARα抑制劑MK886所破壞,此外PPARα專一性啟動劑Wy14643除了能抑制H2O2誘導的細胞凋亡,由此可知此抗細胞凋亡的作用是經由PPARα。同時在VSMC大量表現PPARα會降低H2O2誘導的Caspase 3活化,由此可知PPARα在抵抗細胞凋亡中扮演一個關鍵的角色。緊接著要來確定cPGI2及PPARα啟動劑保護VSMC是經由轉錄調控14-3-3的表現,我們首先分析了7個不同的14-3-3 isoforms,除了σ之外,其餘6個isoform在VSMC內皆持續性的表現,一旦VSMC遭遇氧化壓力,H2O2會降低14-3-3β和θ的蛋白表現量。當使用Caspase 3抑制劑z-DEVD-FMK抑制Caspase 3活性時只有14-3-3β蛋白量恢復,同時在基礎狀態與H2O2作用之下,Ad-COPI及cPGI2亦能增加與恢復14-3-3β蛋白量,使用PPARα啟動劑Wy14643及GW9578大量表現PPARα也能達到相同的效果。當我們大量表現14-3-3β、θ和ε,只有表現14-3-3β具有降低apoptosis的效果,同時使用小分子RNA干擾(small interference RNA)減少14-3-3β的表現量能夠增加Caspase 3的活性。當14-3-3β蛋白量的增加伴隨著促進與Bad(Bcl-2-associated death promoter)蛋白的交互作用,使得Bad蛋白被隔離於細胞質而無法進入粒線體,因此降低apoptosis機制的啟動。綜合以上研究結果得知PPARα所引起14-3-3β蛋白量的增加,在保護細胞抵抗H2O2引起細胞凋亡的機制中扮演一個不可或缺的關鍵角色。


Vascular smooth muscle cell survival is vital to blood vessel integrity. Vascular injury leads to VSMC apoptosis which triggers VSMC migration and proliferation to induce intimal hyperplasia and vascular remodeling. The mechanism by which VSMCs survives under oxidative stress is not entirely clear. We postulated that prostacyclin (PGI2) which is produced by vascular endothelial cells and to a lesser extent by VSMC, protects VSMC against apoptosis by restoring 14-3-3 through a peroxisome proliferator-activated receptor (PPAR)-dependent pathway. We transfected rat VSMCs with an adenoviral vector containing a bicistronic COX-1/PGI synthase construct (Ad-COPI) which co-overexpresses the essential synthetic enzymes (COX-1 and PGI synthase) resulting in robust authentic PGI2 production in VSMC. Ad-COPI transfected cells were less susceptible to apoptosis induced by H2O2. Carbaprostacyclin (cPGI2), a stable analog of PGI2 also attenuated H¬2O2-induced apoptosis. The anti-apoptotic effect of cPGI2 was abrogated by MK 886, a PPARα antagonist and not GSK 3787, a PPARδ antagonist, suggesting that its action is mediated via PPARα. PPARα agonists such as Wy14643 and GW9578 suppressed H2O2-induced apoptosis. Furthermore, PPARα overexpression reduced H2O2-induced caspase 3 activation. These results confirm that PPARα is pivotal in conferring resistance to apoptosis. To determine that PGI2 and PPARα agonists protect VSMC via 14-3-3, we analyzed the protein level of 7 mammalian 14-3-3 isoforms. All isoforms except 14-3-3σ were constitutively expressed. H2O2 treatment resulted in reduction of 14-3- 3β and 14-3-3θ isoforms. 14-3-3β but not 14-3-3θ reduction was rescued by caspase 3 inhibitor, z-DEVD-FMK. Ad-COPI and cPGI2 increased basal 14-3-3β and restored 14-3-3β in H2O2-treated cells. Similarly, Wy14643 as well as PPARα overexpression increased 14-3-3β expression and restored 14-3-3β in H2O2 treated cells. PGI2 and PPARα agonists increase 14-3-3β by transcriptional upregulation. 14-3-3β overexpression attenuated H2O2-induced apoptosis whereas 14-3-3θ or 14-3-3ε overexpression had no effect. Knockdown of 14-3-3β increased VSMC caspase 3 activation. 14-3-3β upregulation was accompanied by enhanced Bad sequestration and reduced Bad translocation to mitochondria. These results reveal that 14-3-3β occupies a central position in oxidant induced apoptosis in VSMCs. The PPARα14-3-3β pathway is a target for developing vascular drugs.
目錄
前言與研究背景
一、血管平滑肌細胞的存活在動脈生理上的重要性 1
二、前列腺素(prostanoids)藉由活化PPARs而保護細胞存活 2
三、Ligand-activated PPARs與14-3-3s在細胞凋亡所扮演之角色 4
材料與方法 8
- 大鼠血管胸動脈平滑肌細胞培養 14
- 西方點墨法 16
- 免疫螢光染色 17
- 即時定量聚合酶連鎖反應 18
- 免疫沉澱 19
- 粒線體萃取 20
- DNA與siRNA 轉染 21
- 腺病毒轉染 21
- 統計分析 22
實驗結果
- H2O2 引發VSMC的apoptosis 23
- PPARα的活化保護VSMC抵抗進入apoptosis 23
- PPARα的overexpression保護VSMC抵抗apoptosis 25
- PPARα的活化使得14-3-3s被upregulate 25
- 14-3-3β保護VSMC抵抗H2O2-induced apoptosis 26
- 活化PPARα隔離Bad進入mitochondria 27
- cPGI2 and Wy14643抗細胞凋亡的效果會被PPAR antagonists破壞 28
討論 30
附圖
圖一 血管的結構與組成 34
圖二 Prostanoids生合成的途徑 35
圖三 PPARs經由調控14-3-3s來保護細胞的存活 36
圖四 H2O2 引發VSMC的apoptosis 37
圖五之一 PPARα及δ的活化保護VSMC避免進入apoptosis 38
圖五之二PPARα agonist抵抗apoptosis存在dose-dependent manner 39
圖五之三 Cleaved caspase 3及nuclei condensation免疫螢光染色 40
圖六 Overexpress PPARα抵抗H2O2 -induced apoptosis 41
圖七之一 活化PPARα的同時能夠upregulate 14-3-3s protein 42
圖七之二 Overexpress PPARα導致14-3-3s protein upregulation 43
圖七之三 PPARα agonist同時會upregulate 14-3-3s mRNA level 44
圖七之四 cPGI2 upregulate 14-3-3β 45
圖七之五 Knockdown 及inhibit PPARα能降低14-3-3β 46
圖七之六 PPARδ無參與調控14-3-3β 47
圖八之一 Overexpress 14-3-3β降低H2O2-induced apoptosis 48
圖八之二 降低14-3-3β增加apoptotic marker 49
圖九 PPARα-upregulated 14-3-3β和Bad結合避免Bad進入mtiochondria 50
圖十PPARα and δ antagonist阻斷cPGI2、Wy14643降低apoptosis 51
圖十一Authentic PGI2 upregulate 14-3-3s且降低apoptosis 52
參考文獻 53
附錄
-附錄一 Ad-COPI construction 65
-附錄二 SDS-PAGE stacking and separating gel preparation 66
-附錄三西方點墨法轉印 67
-附錄四 實驗藥劑配方 68


參考文獻
1. Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev. 1995 Jul;75(3):487-517.
2. Irani K. Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ Res. 2000 Aug 4;87(3):179-83
3. Clarke MC, Figg N, Maguire JJ, Davenport AP, Goddard M, Littlewood TD, Bennett MR. Apoptosis of vascular smooth muscle cells induces features of plaque vulnerability in atherosclerosis. Nat Med. 2006 Sep;12(9):1075-80.
4. Yu H, Clarke MC, Figg N, Littlewood TD, Bennett MR. Smooth muscle cell apoptosis promotes vesselremodeling and repair via activation of cell migration,proliferation, and collagen synthesis. Arterioscler Thromb Vasc Biol. 2011 Nov;31(11):2402-9.
5. Fetalvero, K.M., Martin, K.A. and Hwa, J. Cardioprotective prostacyclin signaling in vascular smooth muscle. Prostaglandins Other Lipid Mediators. 2007, 82: 109-118
6. Flower, R.J. Prostaglandins, bioassay and inflammation. Brit. J. Pharm. 2006, 147: S182-S192
7. Harizi, H., Corcuff, J.B. and Gualde, N. Arachidonic-acid-derived eicosanoids: roles in biology and immunopathology. Trends Mol. Med. 2008, 14: 461-469
8. Parente, l. and Perretti, M. Advances in the pathophysiology of constitutive and inducible cyclooxygenases: two enzymes in the spotlight. Biochem. Pharmacol.2003, 65: 153-159
9. Bingham S, Beswick PJ, Blum DE, Gray NM, Chessell IP. The role of the cyclooxygenase pathway in nociception and pain. Semin Cell Dev Biol. 2006 Oct;17(5):544-54.
10. Forman BM, Chen J, Evans RM. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator activated receptor alpha and delta. Proc Natl Acad Sci. 1997 Apr 29; 94(9):4312-7.
11. Hao CM, Redha R, Morrow J, Breyer MD. Peroxisome proliferator-activated receptor delta activation promotes cell survival following hypertonic stress. J Biol Chem. 2002 Jun 14;277(24):21341-5.
12. Di-Poï N, Tan NS, Michalik L, Wahli W, Desvergne B. Antiapoptotic role of PPARbeta in keratinocytes via transcriptional control of the Akt1 signaling pathway. Mol Cell. 2002 Oct;10(4):721-33.
13. Pesant M, Sueur S, Dutartre P, Tallandier M, Grimaldi PA, Rochette L, Connat JL. Peroxisome proliferator-activated receptor delta (PPARdelta) activation protects H9c2 cardiomyoblasts from oxidative stress-induced apoptosis. Cardiovasc Res. 2006 Feb 1;69(2):440-9.
14. Wan J, Jiang L, Lü Q, Ke L, Li X, Tong N. Activation of PPARdelta up-regulates fatty acid oxidation and energy uncoupling genes of mitochondria and reduces palmitate-induced apoptosis in pancreatic beta-cells. Biochem Biophys Res Commun. 2010 Jan 15;391(3):1567-72
15. Kim HJ, Kim MY, Jin H, Kim HJ, Kang SS, Kim HJ, Lee JH, Chang KC, Hwang JY, Yabe-Nishimura C, Kim JH, Seo HG. Peroxisome proliferator-activated receptor {delta} regulates extracellular matrix and apoptosis of vascular smooth muscle cells through the activation of transforming growth factor-{beta}1/Smad3. Circ Res. 2009 Jul 2;105(1):16-24.
16. Lin H, Lin TN, Cheung WM, Nian GM, Tseng PH, Chen SF, Chen JJ, Shyue SK, Liou JY, Wu CW, Wu KK. Cyclooxygenase-1 and bicistronic cyclooxygenase-1/prostacyclin synthase gene transfer protect against ischemic cerebral infarction. Circulation. 2002 Apr 23;105(16):1962-9.
17. Robinson E, Grieve DJ. Significance of peroxisome proliferator-activated receptors in the cardiovascular system in health and disease. Pharmacol Ther. 2009 Jun;122(3):246-63.
18. Liou JY, Lee S, Ghelani D, Matijevic-Aleksic N, Wu KK. Protection of endothelial survival by peroxisome proliferator-activated receptor-delta mediated 14-3-3 upregulation. Arterioscler Thromb Vasc Biol. 2006 Jul;26(7):1481-7
19. Shyue SK, Tsai MJ, Liou JY, Willerson JT, Wu KK. Selective augmentation of prostacyclin production by combined prostacyclin synthase and cyclooxygenase-1 gene transfer. Circulation. 2001 Apr 24;103(16):2090-5
20. Wu JS, Cheung WM, Tsai YS, Chen YT, Fong WH, Tsai HD, Chen YC, Liou JY, Shyue SK, Chen JJ, Chen YE, Maeda N, Wu KK, Lin TN. Ligand-activated peroxisome proliferator-activated receptor-gamma protects against ischemic cerebral infarction and neuronal apoptosis by 14-3-3 epsilon upregulation. Circulation. 2009 Mar 3;119(8):1124-34.
21. Fu H, Subramanian RR, Masters SC. 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol Toxicol. 2000;40:617-47.
22. Wu KK. Peroxisome Proliferator-Activated Receptors Protect against Apoptosis via 14-3-3. PPAR Res. 2010; 2010. pii: 417646
23. Duriez PJ, Shah GM. Cleavage of poly(ADP-ribose) polymerase: a sensitive parameter to study cell death. Biochem Cell Biol. 1997;75(4):337-49.
24. Liu X, Zou H, Slaughter C, Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell. 1997 Apr 18;89(2):175-84.
25. Liu X, Li P, Widlak P, Zou H, Luo X, Garrard WT, Wang X. The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis. Proc Natl Acad Sci. 1998 Jul 21;95(15):8461-6.
26. Bevan RD. An autoradiographic and pathological study of cellular proliferation in rabbit arteries correlated with an increase in arterial pressure. Blood Vessels. 1976;13(1-2):100-28.
27. Owens GK. Differential effects of antihypertensive drug therapy on vascular smooth muscle cell hypertrophy, hyperploidy, and hyperplasia in the spontaneously hypertensive rat. Circ Res. 1985 Apr;56(4):525-36.
28. Owens GK, Rabinovitch PS, Schwartz SM. Smooth muscle cell hypertrophy versus hyperplasia in hypertension. Proc Natl Acad Sci 1981 Dec;78(12):7759-63.
29. Ross R. The pathogenesis of atherosclerosis--an update. N Engl J Med. 1986 Feb 20;314(8):488-500.
30. Baas AS, Berk BC. Differential activation of mitogen-activated protein kinases by H2O2 and O2- in vascular smooth muscle cells. Circ Res. 1995 Jul;77(1):29-36
31. Bishop-Bailey D, Bystrom J. Emerging roles of peroxisome proliferator-activated receptor-beta/delta in inflammation. Pharmacol Ther. 2009 Nov;124(2):141-50.
32. Kersten S, Desvergne B, Wahli W. Roles of PPARs in health and disease. Nature. 2000 May 25;405(6785):421-4.
33. Hermeking H. The 14-3-3 cancer connection. Nat Rev Cancer. 2003 Dec;3(12):931-43.
34. Morrison DK. The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development. Trends Cell Biol. 2009 Jan;19(1):16-23.
35. Adachi M, Imai K. The proapoptotic BH3-only protein BAD transduces cell death signals independently of its interaction with Bcl-2. Cell Death Differ. 2002 Nov;9(11):1240-7.
36. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell. 1996 Nov 15;87(4):619-28.
37. Wang HG, Rapp UR, Reed JC. Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell. 1996 Nov 15;87(4):629-38.
38. Inohara N, Ding L, Chen S, Núñez G. harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and Bcl-X(L). EMBO J. 1997 Apr 1;16(7):1686-94.
39. del Peso L, González-García M, Page C, Herrera R, Nuñez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science. 1997 Oct 24;278(5338):687-9.
40. Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, Reed JC. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science. 1999 Apr 9;284(5412):339-43.
41. Komatsu K, Miyashita T, Hang H, Hopkins KM, Zheng W, Cuddeback S, Yamada M, Lieberman HB, Wang HG. Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis. Nat Cell Biol. 2000 Jan;2(1):1-6.
42. Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell. 1995 Jan 27;80(2):285-91.
43. Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer. 2002 Apr;2(4):277-88.
44. Hengartner MO. The biochemistry of apoptosis. Nature. 2000 Oct 12;407(6805):770-6.
45. Datta SR, Katsov A, Hu L, Petros A, Fesik SW, Yaffe MB, Greenberg ME. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell. 2000 Jul;6(1):41-51.
46. Nomura M, Shimizu S, Sugiyama T, Narita M, Ito T, Matsuda H, Tsujimoto Y. 14-3-3 Interacts directly with and negatively regulates pro-apoptotic Bax. J Biol Chem. 2003 Jan 17;278(3):2058-65.
47. Walsh K, Smith RC, Kim HS. Vascular cell apoptosis in remodeling, restenosis, and plaque rupture. Circ Res. 2000 Aug 4;87(3):184-8.
48. Hamblin M, Chang L, Fan Y, Zhang J, Chen YE. PPARs and the cardiovascular system. Antioxid Redox Signal. 2009 Jun;11(6):1415-52.
49. Ho TC, Chen SL, Yang YC, Liao CL, Cheng HC, Tsao YP. PEDF induces p53-mediated apoptosis through PPAR gamma signaling in human umbilical vein endothelial cells. Cardiovasc Res. 2007 Nov 1;76(2):213-23.
50. Okura T, Nakamura M, Takata Y, Watanabe S, Kitami Y, Hiwada K. Troglitazone induces apoptosis via the p53 and Gadd45 pathway in vascular smooth muscle cells. Eur J Pharmacol. 2000 Nov 3;407(3):227-35.
51. Obsil T, Ghirlando R, Klein DC, Ganguly S, Dyda F. Crystal structure of the 14-3-3zeta:serotonin N-acetyltransferase complex. a role for scaffolding in enzyme regulation. Cell. 2001 Apr 20;105(2):257-67.
52. Tzivion G, Luo Z, Avruch J. A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature. 1998 Jul 2;394(6688):88-92.
53. Dhillon AS, Meikle S, Peyssonnaux C, Grindlay J, Kaiser C, Steen H, Shaw PE, Mischak H, Eychène A, Kolch W. A Raf-1 mutant that dissociates MEK/extracellular signal-regulated kinase activation from malignant transformation and differentiation but not proliferation. Mol Cell Biol. 2003 Mar;23(6):1983-93.
54. Waterman MJ, Stavridi ES, Waterman JL, Halazonetis TD. ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins. Nat Genet. 1998 Jun;19(2):175-8.
55. Stavridi ES, Chehab NH, Malikzay A, Halazonetis TD. Substitutions that compromise the ionizing radiation-induced association of p53 with 14-3-3 proteins also compromise the ability of p53 to induce cell cycle arrest. Cancer Res. 2001 Oct 1;61(19):7030-3.
56. Liu YC, Liu Y, Elly C, Yoshida H, Lipkowitz S, Altman A. Serine phosphorylation of Cbl induced by phorbol ester enhances its association with 14-3-3 proteins in T cells via a novel serine-rich 14-3-3-binding motif. J Biol Chem. 1997 Apr 11;272(15):9979-85.
57. Craparo A, Freund R, Gustafson TA. 14-3-3ε interacts with the insulin-like growth factor I receptor and insulin receptor substrate I in a phosphoserine-dependent manner. J Biol Chem. 1997 Apr 25;272(17):11663-9.
58. Forrest A, Gabrielli B. Cdc25B activity is regulated by 14-3-3. Oncogene. 2001 Jul 19;20(32):4393-401.
59. Chen MS, Ryan CE, Piwnica-Worms H. Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 bindingCdc25B activity is regulated by 14-3-3. Mol Cell Biol. 2003 Nov;23(21):7488-97.
60. Malik N, Francis SE, Holt CM, Gunn J, Thomas GL, Shepherd L, Chamberlain J, Newman CM, Cumberland DC, Crossman DC. Apoptosis and cell proliferation after porcine coronary angioplasty. Circulation. 1998 Oct 20;98(16):1657-65.
61. Clowes AW, Schwartz SM. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ Res. 1985 Jan;56(1):139-45.
62. Jawien A, Bowen-Pope DF, Lindner V, Schwartz SM, Clowes AW. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest. 1992 Feb;89(2):507-11.
63. Clarke M, Bennett M, Littlewood T. Cell death in the cardiovascular system. Heart. 2007 Jun;93(6):659-64.

連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊